title: A robust and simple-to-design multiplex DNA methylation assay based on MS-MLPA-CE-SSCP.
abstract: Aberrant DNA methylation is a potential diagnostic marker for complex diseases, such as cancer. With the increase in the number of genes known to exhibit disease-associated aberrant methylation, the need for accurate multiplex assays for quantifying DNA methylation has increased. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is one method that has been highlighted in this context. However, two limitations make the custom design of MS-MLPA assays impractical: the need for long probes containing stuffer sequences and a reliance on only one restriction enzyme. Here, we developed a variation of MS-MLPA that employs a simpler probe-design process. To overcome the above-mentioned limitations, we used stuffer-free MS-MLPA probes that are subsequently analyzed using high-resolution capillary electrophoresis-based single-strand conformational polymorphism (CE-SSCP) instead of conventional length-dependent CE. Moreover, multiple methylation-sensitive restriction enzymes (HhaI, HpaII, and AciI) were used simultaneously; thus, probes satisfying desired criteria were available for all targets. Using this assay concept, we analyzed 17 genes associated with hepatocellular carcinoma. Our results showed that the custom-designed assay based on MS-MLPA-CE-SSCP provided robust multiplex quantification of DNA methylation levels.
authors.affiliation: School of Interdisciplinary Bioscience and Bioengineering, Pohang University Science and Technology, Pohang, Gyeongbuk 790-784, Korea. gyjung@postech.ac.kr.
authors.forename: Jeongkyeong
authors.initials: J
authors.lastname: Na
authors.forename: Gi Won
authors.initials: GW
authors.lastname: Shin
authors.forename: Gyu Yong
authors.initials: GY
authors.lastname: Jung
authors.forename: Gyoo Yeol
authors.initials: GY
authors.lastname: Jung
mesh_terms: D001681:Biological Assay / Q000295:instrumentation*; D019175:DNA Methylation; D019075:Electrophoresis, Capillary; D006801:Humans; D013058:Mass Spectrometry; D060885:Multiplex Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational